Inorganic phosphate (Pi) homeostasis is essential for normal development, maintenance, and repair of teeth and skeletal tissues and yet the mechanisms controlling Pi homeostasis remain elusive. Under the parent grant, we hypothesized that cells associated with formation of cementum and dentin, during both development and regeneration, are highly sensitive to changes in Pi homeostasis. For this competitive revision proposal, based on new data suggesting that the ratio of Pi/PPi locally dictates protein expression of bone sialoprotein (BSP) vs. dentin matrix protein-1 (DMP1) in cementum, an additional the hypothesis has been set forth: The ratio of tissue Pi/PPi regulates cementum mineralized tissue apposition as well as composition of cementum extracellular matrix proteins, with an added aim to address this hypothesis: Specific Aim 1;sub-Aim A: To determine the mechanism of Pi/PPi regulation of matrix mineralization and gene/protein expression during tooth root cementogenesis. Strategies include expanding our animal models via a new collaboration with Dr. Jose Luis Millan (Burnham Institute for Medical Research). Dr. Milllan will be providing expertise and also animals with altered Pi/PPi ratio, i.e. Akp2 KO (low Pi/PPi) and Enpp1 KO (high Pi/PPi), and we will be preparing double KO (Akp2;Ank KO and Akp2;Enpp1 KO models). Tissues obtained from these animals will be examined histologically to include analysis of gene/protein expression profiles of single and double KO models by in situ hybridization, immunohistochemistry, and laser capture/microarray. Further, RNA silencing will be used to knock down expression of Ank, Enpp1, and Akp2 in OCCM.30 cementoblasts in vitro to identify, by microarray analyses, factors responding to altered Pi/PPi and potentially regulating BSP/DMP1 expression. The focus will be on identifying Pi/PPi related receptors, transporters, and transcription factors. In addition to providing important basic information on mineralized tissue development, these studies will inform practices relevant to human health. Results from these studies will provide new insights for designing therapies aimed at regenerating periodontal tissues, as well as providing extremely critical knowledge on Pi/PPi metabolism related to maintenance of Pi homeostasis. Furthermore, data generated will provide new information applicable to treatment of Pi associated disorders such as hypophosphatasia (HPP), X-linked hypophosphatemia (XLH), autosomal dominant and recessive hypophospatemic rickets (ADHR, ARHR), familial tumoral calcinosis (FTC), and chronic kidney disease (CKD). All of these severe disorders relate to dysfunction of Pi/PPi homeostasis and feature disorders of bones and teeth. The studies here expand our collaborations outside of dentistry and also will enable us to hire additional staff and a postdoctoral fellow. PUBLIC HEALTH RELEVANCE: Results from these investigations will provide key information as to the modulators of phosphate homeostasis. These new insights will assist in designing therapies aimed at regeneration of periodontal tissues, as well as providing extremely critical knowledge on Pi/PPi metabolism related to disorders such as hypophosphatasia (HPP), X-linked hypophosphatemia (XLH), autosomal dominant and recessive hypophospatemic rickets (ADHR, ARHR), familial tumoral calcinosis (FTC), and chronic kidney disease (CKD). All of these severe disorders relate to dysfunction of Pi/PPi homeostasis and feature disorders of bones and teeth.